MAPPING OF THE RNA-BINDING DOMAIN OF THE CUCUMBER MOSAIC-VIRUS MOVEMENT PROTEIN

A series of in-frame deletion mutants was used to identify a domain wi thin the 3a protein of cucumber mosaic virus (CMV) that is required fo r RNA-binding activity. Deletions in the 3a gene were generated by PCR and restriction digestion, and the resulting mutated 3a sequences wer e cloned either in pT7-7 or in pGEX-5X3 expression vectors. The mutate d 3a proteins or fusions with glutathione S-transferase (GST) were exp ressed in E. coli, purified, and their nucleic acid-binding activities analysed by photochemical UV cross-linking assays using digoxigenin-U TP-labelled RNA probes. Comparative analyses of seven mutated 3a prote ins obtained from inclusion bodies and eight GST fusion proteins revea led that there is an RNA-binding domain located between amino acids 17 4 and 233. This RNA-binding domain is able to bind single-stranded RNA out of the context of the complete 3a movement protein and is highly conserved within both subgroups of CMV.